Ectoine Mitigates Stress and Enhances Lignin to Vanillin Bioconversion in Halalkalibacterium ligniniphilum L1.

Engineered microbial systems offer a route for converting lignin into value-added aromatics, yet lignin-derived intermediates are cytotoxic and suppress growth and metabolic flux. Here, we show that ectoine, a compatible solute, mitigates lignin-induced stress in Halalkalibacterium ligniniphilum L1 and improves bioconversion performance. At 0.1 g L-1, ectoine alleviated surface damage, improved chemical oxygen demand (COD) removal from about 40% to about 55%, and increased decolorization from about 25% to about 48%-50% over 7 days. Assays using crude extracts from recombinant Escherichia coli expressing individual L1-derived enzymes showed that ectoine was associated with broader apparent pH and temperature operating ranges, up to a 12.3% point increase in fixed time substrate conversion, and improved time-dependent retention of measurable activity. Transcriptomic analysis identified 24 differentially expressed genes (9 upregulated and 15 downregulated; false discovery rate (FDR) < 0.05, |log2 fold change (log2 FC) | ≥ 1), indicating a focused rather than global response. Ectoine-responsive changes were associated with membrane/envelope-related functions, transport, and oxidoreductase-related processes, whereas motility and generalized stress-associated programs were reduced. In a 5-L bioreactor, ectoine increased vanillin titers by 13.9%-23.0% at lignin concentrations of 20-40 g L-1. These results support ectoine as an additive for lignin bioconversion under stress.